Journal: Biochemical pharmacology

Article Title: m 6 A modification impacts hepatic drug and lipid metabolism properties by regulating carboxylesterase 2.

doi: 10.1016/j.bcp.2021.114766

Figure Lengend Snippet: Fig. 4. Posttranscriptional regulation of CES2 by RNA methylation via m6A reader protein YTHDC2. The stability of CES2 mRNA in siRNA-transfected HepG2 cells was examined (A-C). HepG2 cells were treated with 10 ng/µL α-amanitin 48 h after transfection with siMETTL3 and siMETTL14 (A), siFTO (B), siALKBH5 (C), or siControl (A-C). Total RNA was prepared after 0, 12, 24, and 36 h. The CES2 mRNA level was determined by using real-time RT-PCR. The CES2 mRNA levels at time 0 (the time of addition of α-amanitin) in each treatment were assigned values of 100%. YTHDC2 mRNA, YTHDF2 mRNA (D), CES2 mRNA (E), and CES2 protein (F) levels in siYTHDC2- or siYTHDF2-transfected HepG2 cells were determined by real-time RT-PCR and Western blotting. The mRNA and protein levels were normalized to β-actin levels. The values represent the levels relative to siControl. (G) Cell lysates from HepG2 cells were immunoprecipitated with an anti-human YTHDC2 antibody or normal rabbit IgG. An electropherogram of the PCR amplicon using primers for CES2 mRNA is shown. The length of the PCR product was 316 bp. Each point and column represent the means ± SD of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.01 compared with siControl.

Article Snippet: The mouse anti-human β-actin monoclonal antibody (sc-37778) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Methylation, Transfection, Quantitative RT-PCR, Western Blot, Immunoprecipitation, Amplification

Journal: Autophagy

Article Title: Resveratrol attenuates vascular endothelial inflammation by inducing autophagy through the cAMP signaling pathway.

doi: 10.4161/auto.26336

Figure Lengend Snippet: Figure 7. RSV-activated SIRT1 via the PRKA-AMPK pathway in endothelial cells. (A) Cells were preincubated with CC (10 μM) or H-89 (10 μM) for 1 h fol- lowed by the addition of RSV for 2 h. In addition, 8-CPT (10 μM) was added for 2 h alone. Then, cells were treated with TNF (10 ng/mL) for another 4 h. The expression of SIRT1, p-AMPK and AMPK was measured by western blot. (B) The bar graphs show the quantification of the indicated proteins. (C) AMPK and (E) PRKA were knocked down by siRNA transfection as described in Materials and Methods. At 24 h post-transfection, the cells were pretreated with RSV (10 μM) for 2 h and then incubated with TNF (10 ng/mL) for an additional 4 h. The cells were collected, lysed and the proteins were subjected to western blot analysis. (D,F) The bar charts show the quantification of the indicated proteins. Values are expressed as means ± SD (n = 3); bP < 0.01 vs. the vehicle-treated control group; dP < 0.01 vs. TNF-treated group; $P < 0.01 vs. RSV and TNF cotreated group; A.U., arbitrary units.

Article Snippet: Intracellular cAMP content in 100-μL aliquots of cell lysate was measured using the cAMP Enzyme Immunoassay Kit, Direct (Sigma-Aldrich, CA200) according to the manufacturer’s instructions. siRNA assay siRNAs for ATG5 (human, sc-41445), BECN1 (human, sc-29797), SIRT1 (human, sc-40986), AMPK (human, sc-45312), PRKA (human, sc-36240) and ADCY (human, sc-43587) were purchased from Santa Cruz Biotechnology along with control siRNA (sc-44230) and siRNA Transfection Reagent (sc-29528).

Techniques: Expressing, Western Blot, Transfection, Incubation, Control

Journal: Autophagy

Article Title: Resveratrol attenuates vascular endothelial inflammation by inducing autophagy through the cAMP signaling pathway.

doi: 10.4161/auto.26336

Figure Lengend Snippet: Figure 8. RSV increased cellular cAMP levels in endothelial cells. (A) Cells were stimulated with or without RSV (10 μM) or forskolin (10 μM) for 2 h, respectively, followed by an additional 4 h incubation in the presence or absence of TNF (10 ng/mL). In addition, cells were treated with KH7 (10 μM) for 1 h or ADCY siRNA transfection following the addition of RSV (10 μM) for 2 h. Then, the cells were incubated with TNF (10 ng/mL) for an additional 4 h. Thereafter, the cells were lysed and 100 μL aliquots of the cleared lysate were used for the cAMP assay as described in Materials and Methods. (B) Cells were preincubated with KH7 (10 μM) for 1 h, followed by the addition of RSV (10 μM) for 2 h; forskolin (10 μM) was added for 2 h. Then, the cells were incu- bated with TNF (10 ng/mL) for an additional 4 h. (C) The bar chart shows the quantification of PRKA. (D) ADCY was knocked down by siRNA transfection in HUVECs as described in Materials and Methods. At 24 h post-transfection, the cells were pretreated with RSV (10 μM) for 2 h and then incubated with TNF (10 ng/mL) for an additional 4 h. The cells were collected, lysed and the proteins were subjected to western blot analysis. (E) The bar charts show the quantification of PRKA. Values are expressed as means ± SD (n = 3); bp < 0.01 vs. the vehicle-treated control group; cP < 0.05, dP < 0.01 vs. TNF-treated cells;

Article Snippet: Intracellular cAMP content in 100-μL aliquots of cell lysate was measured using the cAMP Enzyme Immunoassay Kit, Direct (Sigma-Aldrich, CA200) according to the manufacturer’s instructions. siRNA assay siRNAs for ATG5 (human, sc-41445), BECN1 (human, sc-29797), SIRT1 (human, sc-40986), AMPK (human, sc-45312), PRKA (human, sc-36240) and ADCY (human, sc-43587) were purchased from Santa Cruz Biotechnology along with control siRNA (sc-44230) and siRNA Transfection Reagent (sc-29528).

Techniques: Incubation, Transfection, cAMP Assay, Western Blot, Control

Journal: Autophagy

Article Title: Resveratrol attenuates vascular endothelial inflammation by inducing autophagy through the cAMP signaling pathway.

doi: 10.4161/auto.26336

Figure Lengend Snippet: Figure 9. Regulation of RSV-induced autophagy in endothelial cells. RSV attenuated endothelial inflammation by inducing autophagy via the proposed signaling pathways: RSV increases the cAMP levels possibly through activating ADCY or inhibiting the activity of PDEs, thereby acti- vating cAMP signaling via PRKA and increasing AMPK and SIRT1 activity, and ultimately inducing autophagy.

Article Snippet: Intracellular cAMP content in 100-μL aliquots of cell lysate was measured using the cAMP Enzyme Immunoassay Kit, Direct (Sigma-Aldrich, CA200) according to the manufacturer’s instructions. siRNA assay siRNAs for ATG5 (human, sc-41445), BECN1 (human, sc-29797), SIRT1 (human, sc-40986), AMPK (human, sc-45312), PRKA (human, sc-36240) and ADCY (human, sc-43587) were purchased from Santa Cruz Biotechnology along with control siRNA (sc-44230) and siRNA Transfection Reagent (sc-29528).

Techniques: Protein-Protein interactions, Activity Assay

Journal:

Article Title: Inhibition of Xenopus oocyte meiotic maturation by catalytically inactive protein kinase A

doi: 10.1073/pnas.022056399

Figure Lengend Snippet: Cloning of two Xenopus PKAc isoforms that inhibit oocyte maturation. (A) Identity in the amino acid sequences of XPKAα, XPKAβ, and human PKAc isoforms. (B) Oocytes were injected with the indicated concentrations of mRNAs encoding myc-tagged XPKAα or XPKAβ and incubated overnight before stimulation with progesterone for 20 h. (C) Purified GST-XPKAα (10 ng) and lysates from oocytes untreated (control) and treated with progesterone or injected with mRNAs encoding myc-tagged XPKAα and XPKAβ mRNAs were analyzed by immunoblotting by using the indicated antibodies. (D) The indicated amounts of purified GST-XPKAα and lysates from oocytes either uninjected or injected with XPKAα mRNA were analyzed by immunoblotting with the human PKAγ antibody.

Article Snippet: Immunoblotting was performed as described ( 19 , 21 ) by using the following antibodies: PKAα, PKAβ, PKAγ, PKA-RIIα, myc, p90Rsk1, and p90Rsk2 (Santa Cruz Biotechnology), PKA-RI (Transduction Laboratories, Lexington, KY), phospho-PKA substrate (Cell Signaling Technology, Beverly, MA), Plx1 (provided by D. Glover, University of Cambridge, Cambridge, U.K.), Cdc2 (3E1, provided by J. Gannon and T. Hunt, Imperial Cancer Research Fund, South Mimms, U.K.), p42 MAPK XMpk1 and Myt1 ( 21 ), and XCdc25C (affinity-purified rabbit antiserum prepared against the catalytic domain of Xenopus Cdc25C fused to GST).

Techniques: Clone Assay, Injection, Incubation, Purification, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Histone Deacetylase Inhibitor Belinostat Represses Survivin Expression through Reactivation of Transforming Growth Factor β (TGFβ) Receptor II Leading to Cancer Cell Death

doi: 10.1074/jbc.m110.212035

Figure Lengend Snippet: FIGURE 4. Belinostat treatment induces TGF signaling-dependent survivin down-regulation. Concentration-dependent degradation of survivin mRNA and protein expression in FET (A) and MCF-7L (B) cells treated with belinostat for 48 h is shown. Concentration-dependent inhibition of survivin mRNA and protein expression in MCF-7L cells treated with TSA for 48 h (C) is shown. The mRNA results are expressed as mean S.E. (n 3). Error bars indicate S.E. Western blot analysis was performed to determine the concentration-dependent effects of belinostat (48 h) on survivin in FET and FETDNRII cells (D). Two independent FET cell lines in which Smad2 was knocked down by siRNAs (FETSmad2 siRNA A and B cells, respectively) were treated with belinostat and probed for survivin expression by Western blot (E). Actin was used as a loading control for Western blots. All experiments were performed in triplicate. Cont, control; RQ, relative quantity; Bel, belinostat; pSmad2, phospho-Smad2.

Article Snippet: Anti-actin was purchased from Sigma. shRNAKnockdown Studies—Smad2 shRNA (catalogue number sc-38378-SH) and PKA Cat shRNA (catalogue number sc-36240-SH) were purchased from Santa Cruz Biotechnology, Inc. FET cells were seeded into 10-cm dishes.

Techniques: Concentration Assay, Expressing, Inhibition, Western Blot, Control